Method of site-specific alteration of rna and production of encoded polypeptides

ABSTRACT

A method of site-directed alteration (removal or removal followed by replacement) of selected nucleotides in an RNA molecule, as well as to mixed phosphate backbone oligonucleotides useful in the method. It further relates to a method of producing polypeptides or proteins encoded by the RNA molecule altered by the present method. Through use of the present method, site-directed cleavage of an RNA molecule is effected, followed by excision of the selected or target segment of the RNA molecule.

FUNDING

Work described herein was supported by the National Institutes ofHealth, the G. Harold and Leila Y. Mathers Foundation and the WorcesterFoundation for Experimental Biology.

BACKGROUND

Several aspects of contemporary molecular genetics and biotechnologymake it desirable to be able to produce genetically-altered proteins.For example, mutated protein domains are sometimes hyper-immunogenic,facilitating the production of neutralizing antibody-based vaccines.Moreover, site-directed mutations, ideally one amino acid at a time, canbe a powerful approach to deciphering protein structure and/orenzyme-substrate reaction mechanisms.

Typically, deletions or substitutions of amino acids are made at thegene or DNA level, by recombinant DNA techniques which rely on the useof restriction endonucleases. However, restriction endonucleasesavailable have a limited array of target sites in DNA (usuallypalindromic hexanucleotide or octanucleotide sequences). Deletion of aparticular in-frame trinucleotide or trinucleotides may not be possiblebecause there may be no suitably located restriction sites. As a result,presently-available methods of altering an amino acid sequence byaltering the DNA sequence which encodes it, are limited in theirapplicability.

SUMMARY OF THE INVENTION

The present invention relates to a method of site-directed alteration(removal or removal followed by replacement) of selected nucleotides inan RNA molecule, as well as to mixed phosphate backbone oligonucleotidesuseful in the method. It further relates to a method of producingpolypeptides or proteins encoded by the RNA molecule altered by thepresent method. Through use of the present method, site-directedcleavage of an RNA molecule is effected, followed by excision of theselected or target segment of the RNA molecule. Following cleavage andexcision, in one embodiment, the two segments of the resultinginterrupted RNA molecule are joined, through the action of anappropriate ligase. This results in production of a continuous RNAmolecule referred to as an altered RNA molecule which is the same as theoriginal RNA molecule except that it lacks the nucleotides originallypresent in the target segment of the RNA molecule. In a secondembodiment, selected nucleotides can be introduced into the space or gapcreated by removal of the target RNA segment; a continuous RNA moleculeis created by ligating the selected nucleotides introduced in thismanner to the nucleotide on each side of the gap.

In the present method, an RNA molecule whose nucleotide sequence is tobe altered in a site directed manner is brought into contact with anoligonucleotide, referred as a mixed phosphate backbone oligonucleotide,in the of RNase H. The mixed phosphate backbone oligonucleotide iscomplementary to all or a portion of an RNA molecule which includes atarget segment to be altered. In addition, the mixed phosphate backboneoligonucleotide includes an internal portion or segment ofdeoxynucleotides which is capable of activating RNase H and is flankedon each side by a sequence of nucleotides which is unable to activateRNase H. The internal sequence includes two or more consecutivephosphodiester linkages, which may be unmodified or modified. Theflanking sequences are modified deoxyribonucleotide or ribonucleotidesequences. It has been shown that when such a mixed phosphate backboneoligonucleotide is contacted with a target segment of an RNA molecule,according to the method of the present invention, the result is RNase Hmediated excision of RNA target nucleotides complementary only to theinternal sequence of oligonucleotides. This makes it possible to exciseprecisely any desired nucleotide or oligonucleotide from an RNAmolecule. Followed by RNA ligation, this results in a desired alteredmessenger RNA or other type of RNA Thus, for the first time, it ispossible to carry out precise excision of a selected segment of an RNAmolecule.

As a result, it is possible to selectively delete any desired number ofnucleotides and if desired, to introduce replacement nucleotides. Theencoded amino acid sequence or polypeptide can be produced by expressingthe altered RNA in vitro or in vivo. As a result, a selected amino acidsequence or selected polypeptide can be produced by the present methodby: 1) producing an altered RNA molecule encoding a selected amino acidsequence or selected polypeptide and 2) expressing the altered RNAmolecule under appropriate conditions. Cell-free translation of thealtered RNA molecule can be carried out to produce desired mutantproteins useful, for example, for studies of protein structure orfunction. Alternatively, an appropriate mixed phosphate backboneoligonucleotide can be taken up by or introduced into cultured cells orinto cells of an animal or a plant, in which endogenous RNase H and RNAligase activities can produce altered RNAs; upon translation,corresponding genetically altered proteins are produced. This is useful,for example, as a means of producing defective viral orinfectious/pathogenic agent replication or gene expression, which can beuseful therapeutically or prophylactically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the present method for excisionof a targeted segment of an RNA molecule in which sequence I is RNA fromwhich a targeted segment is to be excised and sequence II is a mixedphosphate backbone oligonucleotide.

Subscript "m" designates an RNase H-resistant internucleoside phosphate;subscript "s" indicates an RNase H-susceptible internucleosidephosphate; "X" designates any of the four ribonucleotides A, C, G or U;and "Y" designates a deoxyribonucleotide complementary to theribonucleotide directly above it.

FIG. 2 is a schematic representation of repair of a genetic defect bythe method of the present invention, in which IVa and IVb represent adouble-stranded hybrid consisting of a smaller unmodified ribonucleotide(IVa) hybridized to a larger modified, RNase H-resistantdeoxyribonucleotide (IVb) and V is the repaired RNA hybridized to theRNase H-resistant deoxyribonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of site-specific orsite-directed alteration (removal or removal followed by replacement) ofselected nucleotides (i.e., a target segment) in an RNA molecule, toproduce an altered RNA sequence, as well as to mixed phosphate backboneoligonucleotides useful in the method. It further relates to a method ofproducing altered amino acid sequences or polypeptides by translatingthe altered RNA sequence, which results in production of the encodedmolecule.

In the present method, a selected or target segment of an RNA molecule,such as pre-mRNA, mRNA or viral RNA, is altered as follows: an RNAmolecule which includes the target segment (i.e., a nucleotide or anucleotide sequence to be altered) is combined with anappropriately-selected mixed phosphate backbone oligonucleotide in thepresence of RNase H. The mixed phosphate backbone oligonucleotide iscomplementary to all or a portion of the RNA molecule which includes thetarget RNA segment; it is of sufficient length to hybridize to thetarget RNA segment and sequences on either side and remain hybridizedunder the conditions used. The mixed phosphate backbone oligonucleotidehas two key components: an internal portion or segment ofdeoxynucleotides which is capable of activating RNase H and twonucleotide sequences, which flank the internal segment, which are unableto activate RNase H. The internal deoxynucleotide segment includes twoor more phosphodiester linkages, which may be unmodified or modified.The flanking nucleotide sequences may be deoxyribonucleotide orribonucleotide sequences and is modified. That is, some or all of theinternucleoside bridging phosphate residues are modified phosphates,such as methyl phosphonates, phosphoromorpholidates,phosphoropiperazidates and phosphoramidates. An essential feature of themixed phosphate backbone oligonucleotide is that the internalcomplementary segment is RNase H activating and the flankingcomplementary sequences are unable to activate RNase H.

As demonstrated herein, site-directed alteration occurs when the RNAmolecule which includes the target segment and an appropriately selectedmixed phosphate backbone oligonucleotide are combined in the presence ofRNase H and maintained under appropriate conditions (e.g., temperature,time, salt concentration) for complementary nucleotide sequences tohybridize and RNase H to be activated (i.e., to be able to cleave andexcise). That is, as a result, the nucleotides in the RNA molecule towhich the internal segment of the mixed phosphate backboneoligonucleotide is complementary are excised precisely from the RNAmolecule.

In one embodiment of the present method, the gap resulting from excisionof the target RNA segment can be closed by the activity of anappropriate ligase(s). resulting in a continuous RNA molecule referredto as an altered RNA molecule. The resulting altered RNA moleculediffers from the RNA molecule only as to the target segment, which isnot present in the altered RNA molecule.

In a second embodiment the gap created by the action of RNase H asdescribed above can be filled in by introducing a segment of replacementnucleotides which can be of any length appropriate to fit into the gapcreated in the RNA molecule. The segment of replacement nucleotides issubsequently linked to the adjacent nucleotides of the RNA molecule byan appropriately selected ligase(s). The nucleotide present at each endof the segment of replacement nucleotides is ligated to the nucleotidepresent on the respective "end" of the gap created in the RNA molecule(underlined in FIG. 2(IIIa). The resulting altered RNA molecule differsfrom the RNA molecule in that the target segment (present in the RNAmolecule) has been removed and a replacement nucleotide sequenceintroduced in its place.

Altered RNA produced as described herein can be expressed, either invitro or in vivo, to produce the encoded polypeptide or protein; as usedherein, the term polypeptide includes proteins. For example, the alteredRNA can be introduced into an appropriate vector, which is in turnintroduced into a host cell capable of translating the altered RNAmolecule and producing the encoded polypeptide. Polypeptides produced inthis manner can be used for assessment of their structural/functionalcharacteristics and a comparative assessment of polypeptides whichdiffer in a defined manner (e.g., by selected amino acids). Suchpolypeptides can also be used therapeutically or prophylactically.

The following is a description, with reference to the figures, of twoembodiments of the present method of site-directed alteration of an RNAmolecule: a first embodiment in which a selected nucleotide sequence(target segment) is removed and the resulting fragments ligated toproduce an altered RNA molecule lacking the target segment and a secondembodiment in which a selected nucleotide sequence is removed andreplaced by a selected nucleotide or nucleotides, which are ligated tothe nucleotide at each side of the gap created by removal of the targetRNA segment.

Method for Excising a Specific Small Segment of Ribonucleotides from anRNA Molecule

FIG. 1 is a schematic representation of an embodiment of the presentmethod. The sequence designated I is a segment of an RNA molecule, whichincludes a target segment (UGACGUCA) to be altered. The RNA may be apre-RNA. a viral RNA, or one of a variety of RNAs present in animal,plant or bacterial cells. The sequence designated II is a segment of DNAwhich is a mixed phosphate backbone oligodeoxynucleotide complementaryto the RNA molecule I. It can be synthesized using known techniques,such as chemical or enzymatic methods. The mixed phosphate backboneoligodeoxynucleotide includes an internal sequence which is capable ofactivating RNase H and is flanked on each side by a DNA segment which isincapable of activating RNase H. The total length of the mixed phosphatebackbone oligodeoxynucleotides varies, depending on the length of thetarget RNA segment to be altered, but must be sufficient to hybridize tothe RNA molecule containing the target RNA segment and remain hybridizedunder the conditions used. The internal segment must be of sufficientlength -- at least two nucleotides -- to be capable of activating RNaseH, as demonstrated herein and by others (Walder, R. Y. and J. A. Walder,Proc. Natl. Acad. Sci. USA, 85:5011-5015 (1988); Furdon, P. J. et al.,Nucleic Acids Res., 17:9193-9204 (1989)).

In the internal segment, the internucleoside bridging phosphate residuesmay be unmodified phosphates or any phosphate modification capable ofactivating RNase H, such as phosphorothioates. The flanking nucleotidesequences can be deoxyribonucleotides, as described in this embodimentor can be ribonucleotides and their modifications. The flankingsequences are connected by methyl phosphonates (PC),phosphoromorpholidates (PM), Phosphoropiperazidates, phosphoramidates,or other modifications of internucleoside phosphates which are not ableto activate RNase H. The RNA molecule I and the modified backboneoligodeoxynucleotide II are combined in the presence of RNase H, such asendogenous RNase H in a cell, and excision of the target segment occurs.A hairpin loop may form by pairing of complementary nucleotides, withthe result that the two newly-formed segments are brought into proximityto each other. It may not be necessary that a hairpin loop form,however, in order for the segments produced by the activity of RNase Hto be brought together because the two RNA segments are tethered bytheir base-pairing with the mixed phosphate backboneoligodeoxynucleotide. In the presence of an appropriate ligase(s). suchas an endogenous ligase(s). the two newly-created segments are joined,by ligation of the nucleotide (underlined in FIG. 1) on each side of thegap.

The definition of "activating RNase H" is based on the induced-fittheory of Koshland, in which "enzyme sites were envisaged as somewhatflexible and undefined before binding occurred, locking active siteresidues into defined positions around the substrate" (Koshland, D. E.,Jr., Proc. Natl. Acad. Sci. USA, 44:98 (1958); Zeffren E. and P. L.Hall, The Study of Enzyme Mechanisms, p. 201, John Wiley and Sons, NewYork, (1973)).

Method for Excising and Replacing a Specific Small Segment ofRibonucleotides from an RNA Molecule

A second embodiment of the present invention is representedschematically in FIG. 2. This embodiment may be useful, as describedbelow, in repairing a genetic defect by removing the defectivenucleotides and replacing them with others, such as those present in thenormal RNA or those which result in RNA encoding a desired polypeptide.As shown, an RNA molecule to be altered, such as pre-mRNA or mRNA inwhich a defect is present, is combined with an appropriate mixedphosphate backbone oligodeoxyribonucleotide in which the internalsegment is capable of activating RNase H and the flanking nucleotidesequences are unable to activate RNase H, in the presence of RNase H. Asdescribed above, the target segment is excised in a site-directedmanner, producing a split RNA molecule, as represented in FIG. 2 assequence IIIa. In this embodiment, a replacement oligomer, which is theseries of nucleotides to be inserted into the gap created by excision ofthe target segment, may be introduced into cells. Through hybridizationexchange (as described in detail below), the replacement oligomer isintroduced into the gap created as a result of the RNase H activity.Expression of the resulting altered RNA (which includes thedesired/nondefective sequence in place of the defective sequence) mayresult in production of the desired/nondefective polypeptide.

Certain genetic disorders (inborn errors of metabolism) may be correctedby the method of the present invention. For example, cystic fibrosis isusually due to a gene mutation in which a specific phenylalanine codonis deleted (Riordon J. R. et al., Science, 245: 1066-1073 (1989)).Through use of the present method, the mutant cystic fibrosis mRNApresent in an individual's cells may be cleaved at the missing codon bysite-directed RNase H alteration. Introduction into the individual'scells of the appropriate oligoribonucleotide (i.e., one encoding aphenylalanine), followed by endogenous RNA ligase activity may result inligation and, thus, production of a wild-type mRNA encoding a normalprotein product.

Other genetic defects may be remedied in a similar manner. Examples ofgenetic defects for which this may be appropriate are: the substitutionof serine for glycine 844 in a severe variant of osteogenesis imperfecta(Pack, M. et al., J. Biol. Chem., 264: 19694-19699 (1989)); a glycine toserine substitution in pro α (II) collagen in a form of dwarfism(Vissing, H. et al., J. Biol. Chem., 264:18265-18267 (1989)); a glycine833 conversion to aspartate in a mild variant of Ehlers-Danlos syndromeIV (Tromp, G. et al., J. Biol. Chem., 264:19313-19317 (1989)); and A toG transition in an initiation codon mutation in the Apo C-II gene of apatient with a deficiency of apolipoprotein C-II (Fojo. S.S. et al., J.Biol. Chem., 264:10839-10842 (1989)); and an aberrant GT splice-donorsignal flanking exon 19 in retinoblastoma RB-88 (Yandell, D. W. et al.,N. E. J. Med., 321:1689-1695 (1989)). Such precise genetic defects maybe repaired at the RNA level by the method described.

Mode of Administration of Modified Backbone Oligonucleotide

The manner in which modified backbone oligonucleotides are provided willdepend on the context in which they are used (i.e., in vitro, in vivo).

The modified backbone oligonucleotide is generally dissolved in water ora suitable buffered medium, such as Dulbecco's medium, Eagle's medium,or a similar physiological saline medium, typically at a concentrationof 10⁻⁵ to 10⁻⁸ molar. In the case of a tissue culture system, thedissolved oligomer is sterilized by filtration through a bacterialfilter, and is added to the other components of the tissue cultureincubation medium. In the case of a seed, the oligomer is dissolved inwater and added to the seeds spread on sterile filter paper inside asterile covered glass or plastic dish. In the case of a plant, dissolvedoligomer, in aqueous media, is added to the soil or other nutrientmaterial in which the plant is growing. In the case of an animal or man,the oligomer, dissolved in physiological saline, may be injectedsubcutaneously, intraperitoneally intra-muscularly, intravenously, orpossibly by capsule orally.

It has been shown that oligomers such as the above-described enterliving cells, and are found in significant concentrations both in thecytoplasm and in the nucleus within minutes after administration(Zamecnik, P. C. et al., Proc. Nat. Acad. Sci. USA, 83:4143-4146 (1986);Goodchild, J. et al., Current Communications in MolecularBiology-Antisense RNA and DNA, Cold Spring Harbor, pp. 135-139 (1988);Wickstrom, E. L. et al., Proc. Nat. Acad. Sci. USA, 85:1028-1032(1988)).

The following exemplification demonstrates that site-specific excisionof nucleotides at the RNA level have been carried out, using the presentmethod and modified backbone oligonucleotides as described herein.

EXEMPLIFICATION Materials and Methods Oligodeoxynucleotide Synthesis

Oligodeoxynucleotides were synthesized on an automated instrument (model8700, Milligen, Mass.). Normal phosphodiester (PO) oligodeoxynucleotidesand the analogous phosphorothioate (PS) or phosphoramidateoligodeoxynucleotides were synthesized using H-phosphonate chemistry(Agrawal, S. et al., Proc. Natl. Acad. Sci. USA, 86:7790-7794 (1989);and Agrawal, S. et al., Proc. Natl. Acad. Sci. USA, 85:7079-7083(1988)). Oligodeoxynucleoside methylphosphonate (PC) analogues wereassembled by using nucleoside methylphosphonamidites (Agrawal, S. and J.Goodchild, Tetrahedron Let., 28: 3539-3542 (1987)).Oligodeoxynucleotides containing both PO and PC internucleoside linkageswere assembled by using nucleoside-β-cyanoethylphosphoramidites andnucleoside methylphosphonamidites, and oligodeoxynucleotides containingboth PC and PS linkages were synthesized from nucleosidemethylphosphonamidites (Agrawal, S. and J. Goodchild, Tetrahedron Lett.,28:3539-3542 (1987)) and nucleoside H-phosphonates.Oligodeoxynucleotides containing both PO and phosphoramidate linkageswere synthesized by using nucleoside-β-cyanoethylphosphoramidites andnucleoside H-phosphonates.

RNase H Assays

The 13-mer oligodeoxynucleotides complementary to nucleotides 2-14 ofhuman U1 small nuclear RNA (counting the G cap as nucleotide (0)) wereadded at 100 μg/ml to a HeLa cell nuclear extract (Dignam, J. D. et al.,Nucleic Acids Res., 11:1475-1479 (1983)) containing 0.5 mM ATP, 20 mMcreatine phosphate, 3.2 mM MgCl₂, and 1000 units of RNase per ml andwere incubated under the conditions specified. RNA was isolated from thenuclear extract by phenol/chloroform extraction and ethanolprecipitation, followed by electrophoresis in a 10% polyacrylamide gelcontaining 8.3M urea. The RNAs were visualized by ethidium bromidestaining.

A second RNase H assay was also carried out in nuclear extracts by usingan exogenous ³² P-labeled RNA. A 514-nucleotide test RNA (hereaftertermed "514 RNA" for convenience) was generated by SP6 RNA polymerasetranscription of a HindIII-linearized pGEM-2 clone, pT7HβΔ6. 514 RNA isantisense to the first two exons and intron of human β-globin pre-mRNAand was chosen for the reasons described below. 514 RNA labeled with a[α˜³² P]GTP, [α˜³² P]CTP, and [α˜³² P]UTP was added to the nuclearextract containing the specified oligodeoxynucleotide at anoligomer-to-514 RNA molar ratio of 3000:1, unless otherwise noted. Afterincubation as specified, RNA was extracted and the ³² P-labeled 514 RNAor its cleavage products were visualized by electrophoresis andautoradiography.

RESULTS Action of RNase H on Oligodeoxynucleotide-U1 Small Nuclear RNAHybrids

HeLa cell nuclear extracts contain RNase H activity that can act onDNA-RNA hybrids that form after addition of oligodeoxynucleotidescomplementary to certain endogenous nuclear RNAs (Krainer, A. R. and T.Maniatis, Cell, 42:725-736 (1985)). The ability of this RNase H activityto cleave the 5'-terminal nucleotides of endogenous U1 small nuclearRNA, after incubation of the various modified oligomers in the nuclearextract, was investigated. Incubation of the PO oligomer in the nuclearextract led to cleavage of a large proportion of the U1 RNA to a product(U1*) having the mobility expected for removal of the first 15nucleotides (cap, nucleotide 1, and nucleotides 2-14). There was a lackof effect on the mobility of any other RNAs present, demonstrating thehigh sequence specificity of the U1 oligomer-directed RNase H cleavage.U1 cleavage was also observed with the PS oligomer, although to aconsistently lesser extent than with the PO oligomer. In contrast, nocleavage was observed with the PC, phosphoroN-morpholidate (PM). orphosphoro-N-butylamidate (PB) oligomers. Reducing the temperature ofincubation to 20° C. and extending the time to 60 minutes did notincrease the extent of cleavage observed with the PS oligomer, nor didit reveal cleavages with the PC, PM or PB oligomers. The pattern of U1cleavages seen with the various oligomers was also not altered by addingE. coli RNase H (Pharmacia, final concentration 8 units/ml) to thenuclear extract at the outset of incubation.

RNase H Action at an Internal RNA Site Hybridized with Normal andModified Oligodeoxynucleotides

The foregoing U1 RNA cleavages involve the 5' extremity of a small RNA.Indeed, the 5' end of U1 RNA is an exceptionally favored target foroligodeoxynucleotide-directed RNase H cleavage since almost all theother regions of this RNA molecule are tightly complexed with proteinsin the U1 small nuclear ribonucleoprotein particle (Patton, J. R. etal., Mol. Cell. Biol., 7: 4030-4037 (1987); Patton. J. R. et al., Proc.Natl. Acad. Sci. USA, 85:747-751 (1988); Patton, J. R. et al., Mol.Cell. Bio., 9:3360-3368 (1989)). Therefore, the action of RNase H onhybrids formed by normal or modified oligomers at internal sites in alonger RNA was also investigated.

For this purpose a test RNA which is 514 nucleotides long (termed 514RNA) was used. 514 RNA is antisense to the first two exons and intron ofhuman β-globin pre-mRNA. The underlying reasoning was that, because itis antisense to a pre-mRNA, 514 RNA would not undergo splicing whenadded to nuclear extract (the cleavage-ligation steps of which wouldotherwise complicate analysis of oligodeoxynucleotide-directed RNase Hcleavages).

A series of normal and modified 15-mer oligodeoxynucleotidescomplementary to nucleotides 349-363 of 514 RNA was synthesizedchemically (Table 1, oligomers A-E). This particular 514 RNA site wasselected because oligomer-directed RNase H cleavage would generate twofragments of readily distinguishable lengths (150 and 348 nucleotides)and also because this 15-nucleotide sequence does not occur elsewhere in514 RNA (Lawn, R. M. et al., Cell, 21:64-651 (1980)). Melting curves in0.16M Na+ for four of these oligodeoxynucleotides after duplex formationwith the complementary (PO) oligomer revealed that the PO, PC, PS and PMoligomer-containing duplexes had t_(m) s of 53° C., 46° C., 43° C. and38° C., respectively, indicating a lower duplex stability for themodified oligomers, in part confirming previous reports (Stein, C. A. etal., Nucleic Acids Res., 16:3209-3221 (1988); Froehler, B. et al.,Nucleic Acids Res., 16:4831-4839 (1988); Quartin, R. S. and J. G.Wetmur, Biochemistry, 28:1040-1047 (1989); Agrawal, S. et al.,Nucleosides Nucleotides, 8:819-823 (1989)).

                  TABLE 1                                                         ______________________________________                                                                     Internucleoside                                  Oligomer                                                                             Sequence              Linkage                                          ______________________________________                                        A      GTA TCA AGG TTA CAA   PO                                               B      GTA TCA AGG TTA CAA   PS                                               C      GTA TCA AGG TTA CAA   PM                                               D      GTA TCA AGG TTA CAA   PB                                               E      GTA TCA AGG TTA CAA   PC                                                F*                                                                                   ##STR1##             PO                                                G*    GTA  .sub.-- GCA AGG  .sub.-- CTA CAA                                                               PO                                                H*                                                                                   ##STR2##             PO                                               ______________________________________                                    

Underlined nucleotides indicate base pairing mismatches; PB isphospho-N-butylamidate.

The series of normal and modified oligodeoxynucleotides shown in Table 1were incubated with ³² P-labeled 514 RNA in nuclear extracts, under theconditions specified in the Materials and Methods section, for either 30minutes or 3.5 hours.

The results of incubating these oligomers with 514 RNA in HeLa nuclearextract are as follows: After 30 minutes of incubation without anyoligomer, intact input 514 RNA was the only labeled species visualized.After 3.5 hours of incubation without any oligomer, the 514 RNA wascompletely degraded by the action of ribonuclease known to be present inthe extract. Incubation of 514 RNA in the extract for 30 minutestogether with either the PO or PS oligomers resulted in precise cleavageof the substrate RNA into two fragments of the sizes expected from thelocation of the oligomer-complementary sequence. Surprisingly, in thecase of the PS oligomer, these two fragments were still present, albeitin slightly degraded form, after 3.5 hours of incubation. Incubation of514 RNA with PM oligomer for 30 minutes resulted in partial cleavage.This partial cleavage is due to the presence of contaminationsunmodified oligomer in the experiment. No cleavage was observed with thePB or PC oligomers under these conditions.

PO oligomers that were only partially complementary to 514 RNA(oligomers F, G and H in Table 1) were also tested. None of theseoligomers, containing 4, 5 or 6 uninterrupted complementary nucleotidesout of the 15 (Table 1), elicited RNase H cleavage of 514 RNA.

The finding that the PS oligomer was less effective than the PO oligomerin eliciting RNase H cleavage in the U1 RNA assay raised the possibilitythat the more complete RNase H cleavages observed with both PO and PSoligomers in the 514 RNA assay might reflect the particular reactionconditions employed. Therefore, a range of oligomer-to-514 RNA molarratios tests (0.1:1-1000:1), all below that used in the above-described(3000:1), was investigated. Results showed that virtually complete 514RNA cleavage occurred with the PO oligomer at an oligomer-to-RNA ratioof 100:1, whereas a comparable extent of 514 cleavage with the PSoligomer occurred at a oligomer-to-RNA ratio of 1000:1. A very similar,incomplete extent of cleavage was observed with the PO and PS oligomersat ratios of 10:1 and 100:1, respectively. The possibility that theseresults might reflect a preferential instability of the PS oligomerduring incubation in the nuclear extracts was examined by experiments inwhich either the PO or the PS oligomer was preincubated in nuclearextract for 30 minutes, followed by addition of ³² P-labeled 514 RNA andincubation for an additional 30 min. This revealed the same extent ofdifference in RNase H cleavage as described above.

"Restriction Endonuclease-Like" Cleavage with Oligomers Containing RNaseH-Sensitive and -Resistant Internucleoside Linkages

The extreme differences between the RNase H sensitivity of DNA-RNAhybrids containing PO or PS oligodeoxynucleotides, contrasted with oneswith PC, PM or PB oligomers, led to an investigation of how RNase H actson a DNA-RNA hybrid in which only a small proportion of internucleosidelinkages in the DNA strand were RNase H-sensitive. Table 2 shows theseries of oligomers that were synthesized to address this issue.

                  TABLE 2                                                         ______________________________________                                        Oligomer    Sequence                                                          ______________________________________                                        I           AGG T                                                             J           A AGG TT                                                                       ##STR3##                                                         L                                                                                          ##STR4##                                                         M                                                                                          ##STR5##                                                         N                                                                                          ##STR6##                                                         O                                                                                          ##STR7##                                                         P                                                                                          ##STR8##                                                         Q                                                                                          ##STR9##                                                         R                                                                                          ##STR10##                                                        ______________________________________                                    

Underlined nucleotides are PC; dashed nucleotides are PS; Boxednucleotides are phosphoromorpholidates; Double-underlined nucleotidesare PM (oligomer Q) or PB; the remaining nucleotides are PO.

The oligomers listed were tested for their capacity to elicit RNase Haction after hybridization to 514 RNA, as in the preceding experiments.

Results demonstrated that neither a tetramer nor a hexamer (all PO)oligodeoxynucleotide complementary to 514 RNA was able to induce RNase Hcleavage in this nuclear extract system. When PO/PC-containingpentadecamers containing either two or four consecutive PO linkages wereused, a low but readily detectable level of RNase H cleavage occurred.In contrast, a pendtadecamer containing six consecutive PO linkageselicited complete RNase H cleavage of the substrate RNA. Note that thesix PO nucleotides in this oligomer (oligomer M in Table 2) areidentical in sequence to the RNase H-inactive hexamer (oligomer J), fromwhich it is inferred that the potency of the pentadecamer reflects itsincreased hybrid stability with 514 RNA owing to the additional ninecomplementary nucleotides.

Similar tests were performed with mixed PO/PC pentadecamerscomplementary to a different site in 514 RNA (i.e., nucleotides463-477). These tests revealed an effect of the number of PO linkages onRNase H cleavage similar to that described above.

Additional variants of mixed PO/PC oligomers were also tested.Pentadecamer PO/PC oligomers with five or six consecutive PO linkages ateither the extreme 5' or 3' end were highly effective in eliciting RNaseH cleavage. A PS/PC pentadecamer with six consecutive PS linkages at theextreme 5' end (oligomer P in Table 2) was only partially active.Comparison of these results with lanes 2 and 3 in FIG. 4B confirms theabove-described results showing that all-PS oligomers are less effectivethan all-PO oligomers in eliciting RNase H cleavage. RNase H cleavage of514 RNA was also observed with a 15-mer containing nine consecutive PMor PB linkages followed by six PO-linked nucleotides (oligomers Q and Rin Table 2).

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed by the scope of the followingclaims.

We claim:
 1. A mixed phosphate backbone oligonucleotide consistingessentially of an internal segment of modified nucleotides whichactivates RNase H and two modified nucleotide sequences which do notactivate RNAse H, in which the two modified nucleotide sequences flankthe internal segments, one on each side of the internal segment, whereinthe internucleoside bridging phosphate residues of the internal segmentare modified phosphates which are phosphorothioates and theinternucleoside bridging phosphate residues of the two flanking modifiednucleotide sequences are modified phosphate selected from the groupconsisting of: methyl phosphonates, phosphoromorpholidates,phosphoropiperazidates, and phosphoramidates.
 2. The mixed phosphatebackbone oligonucleotide of claim 1, wherein the internucleosidebridging phosphate residues of the two flanking modified nucleotidesequences are methyl phosphonates.
 3. The mixed phosphate backboneoligonucleotide of claim 1, wherein the internucleoside bridgingphosphate residues of the two flanking modified nucleotide sequences arephosphoromorpholidates.
 4. The mixed phosphate backbone oligonucleotideof claim 1, wherein the internucleoside bridging phosphate residues ofthe two flanking modified nucleotide sequences arephosphoropiperazidates.
 5. The mixed phosphate backbone oligonucleotideof claim 1, wherein the internucleoside bridging phosphate residues ofthe two flanking modified nucleotide sequences are phosphoramidates.